| Objective:To study the chondrogenic differentiation of BMSCs infected with Ad-TGF?1attached to 3D-printed PLA scaffolds,providing experimental basis for in vivo repair of articular cartilage defects in rabbits with BMSC-PLA composite scaffolds.Methods:1.2 months old New Zealand white rabbits were sacrificed for hypoxia.BMSCs were isolated from femur and tibia of both hind limbs and cultured every 2-3 days.2.Ad-TGF?1 was amplified and the titer of Ad-TGF?1 was detected after amplification.The amplified Ad-TGF?1 was used to infect BMSCs cells,and the TGF beta1 gene was successfully introduced into BMSCs cells.In the same way,the empty vector of Ad-TGF?1 was used to infect BMSCs cells,and the empty vector of Ad-TGF?1 was introduced into BMSCs for backup.At day 7,the number of green fluorescent cells was observed with a fluorescence microscope under a 100-fold field of view,and the infection efficiency was calculated.3.Rabbit knee cartilage defect model was made,and CT scan was performed to collect the original data of cartilage defect.PLA was used as the printing material to design scaffolds suitable for aperture size.Sterilize and reserve.4.Eighteen PLA scaffolds were taken and six PLA scaffolds were randomly co-cultured with BMSCs of the Ad-TGF?1 infected group,another six PLA scaffolds were randomly co-cultured with BMSCs of the no-load group,and the remaining six PLA scaffolds were co-cultured with BMSCs of the blank control group.The solution was changed every 2-3 days,and the adhesion rate of each group of cells on the PLA scaffold was determined by cell counting method.Cell adhesion was observed by sem after 5days.CCK8 detected the cell activity of each group on PLA scaffold.Take each compound support cells into cartilage induced,after 7 days of toluidine blue staining detection cartilage cell differentiation,q PCR detection relative expression of TGF beta 1 m RNA,Western blot detection COL-? expression;SPSS22.0 software was used for statistical analysis of data.Measurement data were normally distributed and represented by mean ± standard deviation(±s).Anova was used for pairwise comparison between three groups and more,and LSD test was used for pairwise comparison.Results: 1.BMSCs were successfully isolated from rabbit bone marrow.2.After the constructed Ad-TGF?1 infected HEK293 cells,the CEP phenomenon was obvious,and the titer of Ad-TGF?1 virus was detected as 5xl08PFU/m L,which was successfully amplified.The amplified Ad-TGF?1 was infected with BMSCs with an infection efficiency of 80%.3.The 100 um PLA cartilage defect stent with pore size was prepared by 3D printing technology.4.Electron microscope scanning showed that there was cell adhesion on the surface of PLA scaffolds in all groups,and linear protrusions were found to be extended and attached to the scaffolds.However,there was significant difference in cell adhesion rate between the cocultured groups of BMSCs and PLA scaffolds,and the comparison between the groups with the highest cell adhesion rate in the transfection group was statistically significant(P < 0.05).The relative expression of TGF ? 1m RNA in each group was detected on the 7th day of chondrogenic induction of scaffold cell complex.The relative expression of TGF ? 1m RNA in the transfection group was 2.28±0.13,significantly higher than that in the blank group and no-load group,with statistically significant difference(P <0.05).Toluidine blue staining showed blue-purple nuclei in the infected group and negative toluidine blue staining in the no-load group and the blank group.Electrophoresis results showed stripe dyed deep infection group,COL-? relative expression was 1.40 ± 0.06,the blank group and high light group,the difference was statistically significant(P < 0.05).Conclusion:1.Surface modification of 3d-printed PLA scaffolds can enhance the adhesion properties of Ad-TGF?1 infected BMSCs PLA scaffolds.2.Ad-TGF?1 induced chondrocyte differentiation by BMSCs adhesion to3d-printed PLA scaffolds. |
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